Ureaplasma Parvum Serovar Research Articles

Repeated Intrauterine Exposures to Inflammatory Stimuli Attenuated Transforming Growth Factor-β Signaling in the Ovine Fetal Lung

Background: Bronchopulmonary dysplasia (BPD) is one of the most common complications after preterm birth and is associated with intrauterine exposure to bacteria. Transforming growth factor-β (TGFβ) is implicated in the development of BPD. Objectives: We hypothesized that different and/or multiple bacterial signals could elicit divergent TGFβ signaling responses in the developing lung. Methods: Time-mated pregnant Merino ewes received an intra-amniotic injection of lipopolysaccharide (LPS) and/or Ureaplasma parvum serovar 3 (UP) at 117 days' and/or 121/122 days' gestational age (GA). Controls received an equivalent injection of saline and or media. Lambs were euthanized at 124 days' GA (term = 150 days' GA). TGFβ1, TGFβ2, TGFβ3, TGFβ receptor (R)1 and TGFβR2 protein levels, Smad2 phosphorylation and elastin deposition were evaluated in lung tissue. Results: Total TGFβ1 and TGFβ2 decreased by 24 and 51% after combined UP+LPS exposure, whereas total TGFβ1 increased by 31% after 7 days' LPS exposure but not after double exposures. Alveolar expression of TGFβR2 decreased 75% after UP, but remained unaltered after double exposures. Decreased focal elastin deposition after single LPS exposure was prevented by double exposures. Conclusions: TGFβ signaling components and elastin responded differently to intrauterine LPS and UP exposure. Multiple bacterial exposures attenuated TGFβ signaling and normalized elastin deposition.

Ureaplasma parvum Serovar 3 Multiple Banded Antigen Size Variation after Chronic Intra-Amniotic Infection/Colonization

Ureaplasma species are the microorganisms most frequently associated with adverse pregnancy outcomes. The multiple banded antigen (MBA), a surface-exposed lipoprotein, is a key virulence factor of ureaplasmas. The MBA demonstrates size variation, which we have shown previously to be correlated with the severity of chorioamnion inflammation. We aimed to investigate U. parvum serovar 3 pathogenesis in vivo, using a sheep model, by investigating: MBA variation after long term (chronic) and short term (acute) durations of in utero ureaplasma infections, and the severity of chorioamnionitis and inflammation in other fetal tissues. Inocula of 2×107 colony-forming-units (CFU) of U. parvum serovar 3 (Up) or media controls (C) were injected intra-amniotically into pregnant ewes at one of three time points: day 55 (69d Up, n = 8; C69, n = 4); day 117 (7d Up, n = 8; C7, n = 2); and day 121 (3d Up, n = 8; C3, n = 2) of gestation (term = 145–150d). At day 124, preterm fetuses were delivered surgically. Samples of chorioamnion, fetal lung, and umbilical cord were: (i) snap frozen for subsequent ureaplasma culture, and (ii) fixed, embedded, sectioned and stained by haematoxylin and eosin stain for histological analysis. Selected fetal lung clinical ureaplasma isolates were cloned and filtered to obtain cultures from a single CFU. Passage 1 and clone 2 ureaplasma cultures were tested by western blot to demonstrate MBA variation. In acute durations of ureaplasma infection no MBA variants (3d Up) or very few MBA variants (7d Up) were present when compared to the original inoculum. However, numerous MBA size variants were generated in vivo (alike within contiguous tissues, amniotic fluid and fetal lung, but different variants were present within chorioamnion), during chronic, 69d exposure to ureaplasma infection. For the first time we have shown that the degree of ureaplasma MBA variation in vivo increased with the duration of gestation.

Synergic Activation of Toll-Like Receptor (TLR) 2/6 and 9 in Response to Ureaplasma parvum & urealyticum in Human Amniotic Epithelial Cells

Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to “sense” pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.

Interaction of the putative tyrosine recombinases RipX (UU145), XerC (UU222), and CodV (UU529) ofUreaplasma parvumserovar 3 with specific DNA

Phase variation of two loci (‘mba locus’ and ‘UU172 phase-variable element’) in Ureaplasma parvum serovar 3 has been suggested as result of site-specific DNA inversion occurring at short inverted repeats. Three potential tyrosine recombinases (RipX, XerC, and CodV encoded by the genes UU145, UU222, and UU529) have been annotated in the genome of U. parvum serovar 3, which could be mediators in the proposed recombination event. We document that only orthologs of the gene xerC are present in all strains that show phase variation in the two loci. We demonstrate in vitro binding of recombinant maltose-binding protein fusions of XerC to the inverted repeats of the phase-variable loci, of RipX to a direct repeat that flanks a 20-kbp region, which has been proposed as putative pathogenicity island, and of CodV to a putative dif site. Co-transformation of the model organism Mycoplasma pneumoniae M129 with both the ‘mba locus’ and the recombinase gene xerC behind an active promoter region resulted in DNA inversion in the ‘mba locus’. Results suggest that XerC of U. parvum serovar 3 is a mediator in the proposed DNA inversion event of the two phase-variable loci.

Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvumstrains

BackgroundUreaplasma urealyticum (UUR) and Ureaplasma parvum (UPA) are sexually transmitted bacteria among humans implicated in a variety of disease states including but not limited to: nongonococcal urethritis, infertility, adverse pregnancy outcomes, chorioamnionitis, and bronchopulmonary dysplasia in neonates. There are 10 distinct serotypes of UUR and 4 of UPA. Efforts to determine whether difference in pathogenic potential exists at the ureaplasma serovar level have been hampered by limitations of antibody-based typing methods, multiple cross-reactions and poor discriminating capacity in clinical samples containing two or more serovars.ResultsWe determined the genome sequences of the American Type Culture Collection (ATCC) type strains of all UUR and UPA serovars as well as four clinical isolates of UUR for which we were not able to determine serovar designation. UPA serovars had 0.75−0.78 Mbp genomes and UUR serovars were 0.84−0.95 Mbp. The original classification of ureaplasma isolates into distinct serovars was largely based on differences in the major ureaplasma surface antigen called the multiple banded antigen (MBA) and reactions of human and animal sera to the organisms. Whole genome analysis of the 14 serovars and the 4 clinical isolates showed the mba gene was part of a large superfamily, which is a phase variable gene system, and that some serovars have identical sets of mba genes. Most of the differences among serovars are hypothetical genes, and in general the two species and 14 serovars are extremely similar at the genome level.ConclusionsComparative genome analysis suggests UUR is more capable of acquiring genes horizontally, which may contribute to its greater virulence for some conditions. The overwhelming evidence of extensive horizontal gene transfer among these organisms from our previous studies combined with our comparative analysis indicates that ureaplasmas exist as quasi-species rather than as stable serovars in their native environment. Therefore, differential pathogenicity and clinical outcome of a ureaplasmal infection is most likely not on the serovar level, but rather may be due to the presence or absence of potential pathogenicity factors in an individual ureaplasma clinical isolate and/or patient to patient differences in terms of autoimmunity and microbiome.

Inflammation of the Fetal Ovine Skin Following in utero Exposure to Ureaplasma parvum

There is increasing evidence linking in utero infection and inflammation to preterm birth. Many commensal urogenital tract microorganisms, including the Mycoplasmas and Ureaplasmas, are commonly detected in association with preterm birth. Using an ovine model of sterile fetal inflammation, we demonstrated previously that the fetal skin generates a robust inflammatory response following in utero exposure to lipopolysaccharides from Escherichia coli. The fetal skin's response to colonization of the amniotic fluid by viable microorganisms remains unstudied. We hypothesised that in utero infection with Ureaplasma parvum serovar 3 would induce a proinflammatory response in the fetal skin. We found that (1) cultured fetal keratinocytes (the primary cellular constituent of the epidermis) respond to U. parvum exposure in vitro by increasing the expression of the chemotactant monocyte chemoattractant protein 1 (MCP-1) but not interleukin 1β (IL-1β), IL-6, IL-8, or tumor necrosis factor-α (TNF-α); (2) the fetal skin's response to 7 days of U. parvum exposure is characterized by elevated expression of MCP-1, TNF-α, and IL-10; and (3) the magnitude of inflammatory cytokine/chemokine expression in the fetal skin is dependent on the duration of U parvum exposure. These novel findings provide further support for the role of the fetal skin in the development of fetal inflammation and the preterm birth that may follow.

Chronic Fetal Exposure to Ureaplasma parvum Suppresses Innate Immune Responses in Sheep

The chorioamnionitis associated with preterm delivery is often polymicrobial with ureaplasma being the most common isolate. To evaluate interactions between the different proinflammatory mediators, we hypothesized that ureaplasma exposure would increase fetal responsiveness to LPS. Fetal sheep were given intra-amniotic (IA) injections of media (control) or Ureaplasma parvum serovar 3 either 7 or 70 d before preterm delivery. Another group received an IA injection of Escherichia coli LPS 2 d prior to delivery. To test for interactions, IA U. parvum-exposed animals were challenged with IA LPS and delivered 2 d later. All animals were delivered at 124 ± 1-d gestation (term = 150 d). Compared with the 2-d LPS exposure group, the U. parvum 70 d + LPS group had 1) decreased lung pro- and anti-inflammatory cytokine expression and 2) fewer CD3(+) T lymphocytes, CCL2(+), myeloperoxidase(+), and PU.1(+) cells in the lung. Interestingly, exposure to U. parvum for 7 d did not change responses to a subsequent IA LPS challenge, and exposure to IA U. parvum alone induced mild lung inflammation. Exposure to U. parvum increased pulmonary TGF-β1 expression but did not change mRNA expression of either the receptor TLR4 or some of the downstream mediators in the lung. Monocytes from fetal blood and lung isolated from U. parvum 70 d + LPS but not U. parvum 7 d + LPS animals had decreased in vitro responsiveness to LPS. These results are consistent with the novel finding of downregulation of LPS responses by chronic but not acute fetal exposures to U. parvum. The findings increase our understanding of how chorioamnionitis-exposed preterm infants may respond to lung injury and postnatal nosocomial infections.

Ureaplasma antigenic variation beyond MBA phase variation: DNA inversions generating chimeric structures and switching in expression of the MBA N-terminal paralogue UU172

Phase variation of the major ureaplasma surface membrane protein, the multiple-banded antigen (MBA), with its counterpart, the UU376 protein, was recently discussed as a result of DNA inversion occurring at specific inverted repeats. Two similar inverted repeats to the ones within the mba locus were found in the genome of Ureaplasma parvum serovar 3; one within the MBA N-terminal paralogue UU172 and another in the adjacent intergenic spacer region. In this report, we demonstrate on both genomic and protein level that DNA inversion at these inverted repeats leads to alternating expression between UU172 and the neighbouring conserved hypothetical ORF UU171. Sequence analysis of this phase-variable ‘UU172 element’ from both U. parvum and U. urealyticum strains revealed that it is highly conserved among both species and that it also includes the orthologue of UU144. A third inverted repeat region in UU144 is proposed to serve as an additional potential inversion site from which chimeric genes can evolve. Our results indicate that site-specific recombination events in the genome of U. parvum serovar 3 are dynamic and frequent, leading to a broad spectrum of antigenic variation by which the organism may evade host immune responses.

Inflammation in fetal sheep from intra-amniotic injection of Ureaplasma parvum

Bronchopulmonary dysplasia is associated with chorioamnionitis and fetal lung inflammation. Ureaplasma species are the bacteria most frequently isolated from chorioamnionitis. Very chronic ureaplasma colonization of amniotic fluid causes low-grade lung inflammation and functional lung maturation in fetal sheep. Less is known about shorter exposures of the fetal lung. Therefore, we hypothesized that ureaplasmas would cause an acute inflammatory response that would alter lung development. Singleton ovine fetuses received intra-amniotic Ureaplasma parvum serovar 3 or control media at 110, 117, or 121 days and were delivered at 124 days gestational age (term = 150 days). Inflammation was assessed by 1) cell counts in bronchoalveolar lavage fluid (BALF), and 2) cytokine mRNA measurements, immunohistochemistry, and flow cytometry for inflammatory cells and elastin and α-smooth muscle actin (α-SMA) staining in lung tissue. Neutrophils were increased in BALF 3 days after exposure to ureaplasmas (P = 0.01). Myeloperoxidase-positive cells increased after 3 days (P = 0.03), and major histocompatibility complex (MHC) class II-positive cells increased after 14 days of ureaplasma exposure (P = 0.001). PU.1 (macrophage marker)- or CD3 (T lymphocyte marker)-positive cells were not induced by ureaplasmas. CD3-positive cells in the posterior mediastinal lymph node increased in ureaplasma-exposed animals at 3, 7, and 14 days (P = 0.002). Focal elastin depositions decreased in alveolar septa at 14 days (P = 0.002), whereas α-SMA increased in arteries and bronchioli. U. parvum induced a mild acute inflammatory response and changed elastin and α-SMA deposition in the lung, which may affect lung structure and subsequent development.

Maternal Administration of Erythromycin Fails to Eradicate Intrauterine Ureaplasma Infection in an Ovine Model1

Erythromycin is the standard antibiotic used for treatment of infection with Ureaplasma spp. during pregnancy; however, maternally administered erythromycin may be ineffective at eliminating intra-amniotic ureaplasma infections. We examined whether erythromycin would eradicate intra-amniotic ureaplasma infections in pregnant sheep. At Gestational Day (GD) 50 (term, GD 150), pregnant ewes received intra-amniotic injections of erythromycin-sensitive Ureaplasma parvum serovar 3 (n = 16) or 10B medium (n = 16). At GD 100, amniocentesis was performed; five fetal losses (ureaplasma group, n = 4; 10B group, n = 1) had occurred by this time. Remaining ewes were allocated into treatment subgroups: medium only (n = 7), medium and erythromycin (n = 8), ureaplasma only (Up; n = 6), or ureaplasma and erythromycin (Up/E; n = 6). Erythromycin was administered intramuscularly (500 mg) every 8 h for 4 days (GDs 100-104). Amniotic fluid samples were collected at GD 105. At GD 125, preterm fetuses were surgically delivered, and specimens were collected for culture and histology. Erythromycin was quantified in amniotic fluid by liquid chromatography-mass spectrometry. Ureaplasmas were isolated from the amniotic fluid, chorioamnion, and fetal lung of animals from the Up and Up/E groups, however, the numbers of U. parvum recovered were not different between these groups. Inflammation in the chorioamnion, cord, and fetal lung was increased in ureaplasma-exposed animals compared to controls but was not different between the Up and Up/E groups. Erythromycin was detected in amniotic fluid samples, although concentrations were low (<10-76 ng/ml). This study demonstrates that maternally administered erythromycin does not eradicate chronic, intra-amniotic ureaplasma infections or improve fetal outcomes in an ovine model, potentially because of the poor placental passage of erythromycin.

The Severity of Chorioamnionitis in Pregnant Sheep Is Associated with In Vivo Variation of the Surface-Exposed Multiple-Banded Antigen/Gene of Ureaplasma parvum1

Ureaplasma species are the bacteria most frequently isolated from human amniotic fluid in asymptomatic pregnancies and placental infections. Ureaplasma parvum serovars 3 and 6 are the most prevalent serovars isolated from men and women. We hypothesized that the effects on the fetus and chorioamnion of chronic ureaplasma infection in amniotic fluid are dependent on the serovar, dose, and variation of the ureaplasma multiple-banded antigen (MBA) and mba gene. We injected high- or low-dose U. parvum serovar 3, serovar 6, or vehicle intra-amniotically into pregnant ewes at 55 days of gestation (term = 150 days) and examined the chorioamnion, amniotic fluid, and fetal lung tissue of animals delivered by cesarean section at 125 days of gestation. Variation of the multiple banded antigen/mba generated by serovar 3 and serovar 6 ureaplasmas in vivo were compared by PCR assay and Western blot. Ureaplasma inoculums demonstrated only one (serovar 3) or two (serovar 6) MBA variants in vitro, but numerous antigenic variants were generated in vivo: serovar 6 passage 1 amniotic fluid cultures contained more MBA size variants than serovar 3 (P = 0.005), and ureaplasma titers were inversely related to the number of variants (P = 0.025). The severity of chorioamnionitis varied between animals. Low numbers of mba size variants (five or fewer) within amniotic fluid were associated with severe inflammation, whereas the chorioamnion from animals with nine or more mba variants showed little or no inflammation. These differences in chorioamnion inflammation may explain why not all women with in utero Ureaplasma spp. experience adverse pregnancy outcomes.

Ventilation-Mediated Injury After Preterm Delivery of Ureaplasma parvum Colonized Fetal Lambs

Ureaplasma species are the microorganisms most frequently isolated from women with preterm birth and are associated with an increased risk of bronchopulmonary dysplasia. Initiation of ventilation with high tidal volumes (VT) causes lung injury and inflammation. We investigated whether antenatal colonization with Ureaplasma parvum serovar 3 (UP) would alter the inflammatory response to mechanical ventilation of preterm lambs. Merino ewes were given intraamniotic injections of UP at 55-d gestation, and the lambs were surgically delivered at 128+/-1 d gestation and assigned to three groups: 1) gentle ventilation (GV), 2) high VT ventilation, or 3) unventilated control. Lambs delivered from noncolonized ewes were assigned to parallel groups. GV lambs received surfactant before ventilation with a VT of 7 mL/kg, positive end expiratory pressure (PEEP) 5 cm H2O. High VT lambs received no PEEP and escalating VT to 15 mL/kg by 15 min. At 15 min, surfactant was given, VT was reduced to 7 mL/kg, and PEEP was increased to 5 cm H2O. Monocytes in bronchoalveolar lavage were increased by UP, but colonization did not affect lung function. High VT ventilation increased Egr-1 signaling, proinflammatory cytokine expression, and injury scores compared with GV. Antenatal colonization with UP did not change lung function or modulate the lung injury and inflammation caused by high VT ventilation.

Pulmonary vascular and alveolar development in preterm lambs chronically colonized with Ureaplasma parvum.

Ureaplasma species, the most commonly isolated microorganisms in women with chorioamnionitis, are associated with preterm delivery. Chorioamnionitis increases the risk and severity of bronchopulmonary dysplasia and persistent pulmonary hypertension in newborns. It is not known whether the timing of exposure to inflammation in utero is an important contributor to the pathogenesis of bronchopulmonary dysplasia. We hypothesized that chronic inflammation would alter the pulmonary air space and vascular development after 70 days of exposure to infection. Pregnant ewes were given intra-amniotic injection of Ureaplasma parvum serovars 3 or 6 at low (2 x 10(4) cfu) or high doses (2 x 10(7) cfu) or media (controls) at 55 days gestational age. Fetuses were delivered at 125 days (term = 150 days). U. parvum was grown from the lungs of all exposed fetuses, and neutrophils and monocytes were increased in the air spaces. Lung mRNA expression of IL-1beta and IL-8, but not IL-6, was modestly increased in U. parvum-exposed fetuses. U. parvum exposure increased surfactant and improved lung gas volumes. The changes in lung inflammation and maturation were independent of serovar or dose. Exposure to U. parvum did not change multiple indices of air space or vascular development. Parenchymal elastin and collagen content were similar between groups. Expression of several endothelial proteins and pulmonary resistance arteriolar media thickness were also not different between groups. We conclude that chronic exposure to U. parvum does not cause sustained effects on air space or vascular development in premature lambs.

Comparison of full gyrA, gyrB, parC and parE gene sequences between all Ureaplasma parvum and Ureaplasma urealyticum serovars to separate true fluoroquinolone antibiotic resistance mutations from non-resistance polymorphism

To determine the role of amino acid substitutions in Ureaplasma GyrA, GyrB, ParC and ParE proteins in mediating fluoroquinolone resistance. Nucleic acid sequences from gyrA, gyrB, parC and parE genes from all 14 Ureaplasma serovars were aligned. Full genome sequences for serovars 1, 3-7, 9 and 11-14 were available from the National Center for Biotechnology Information database and we sequenced the full topoisomerase genes from ciprofloxacin-susceptible reference strains of serovars 2, 8 and 10. Phylogenetic trees were constructed to analyse nucleotide sequence similarity. Deduced amino acid sequences were compared with all 33 previously reported fluoroquinolone-resistant strains to clarify true fluoroquinolone-resistance-associated substitutions. Non-resistance-associated polymorphisms were identified in GyrA (39), GyrB (26), ParC (107) and ParE (34) proteins. Phylogenetic analysis demonstrated species clustering for all genes, except parE in which serovars 4, 12, 10 and 13 formed a separate cluster more similar to Ureaplasma parvum than the remaining Ureaplasma urealyticum serovars. Examination of all previously reported fluoroquinolone-resistant strains found that one-third of identified residue substitutions could be attributed to normal species polymorphism; therefore, the mechanism of resistance for these strains is still undetermined. In particular, Glu or Asp at position 112 in GyrA and Ala or Thr at 125/136 in ParC were substitutions identified when U. urealyticum strain sequences were previously aligned with the published serovar 3 genome sequence. Combining analysis of the recently available Ureaplasma genomes with sequences from the additional serovars has enabled us to clarify which substitutions found by previous investigators could potentially be responsible for fluoroquinolone resistance.

Ureaplasma colonization of amniotic fluid and efficacy of antenatal corticosteroids for preterm lung maturation in sheep

The objective of the study was to assess the efficacy of maternal betamethasone for improving preterm lung function in the presence of inflammation induced by amniotic fluid Ureaplasma colonization. Ewes bearing single fetuses were randomized to receive an intraamniotic injection of Ureaplasma parvum (serovar 6; 2 x 10(7) colony-forming units) or vehicle at 86 +/- 2 days of pregnancy (mean +/- SD: term is 150 days), followed by maternal intramuscular betamethasone (0.5 mg/kg) or saline, either 2 or 7 days before delivery of lambs at 123 +/- 1 d. Amniotic fluid interleukin-8 was elevated by ureaplasmas (P = .049) but unaffected by betamethasone. Lung inflammation induced by ureaplasmas was not affected by betamethasone. Lung compliance was increased by Ureaplasma colonization (P = .009) and betamethasone (P = .042), and effects were additive. Lung surfactant was increased by Ureaplasma colonization (P < .001) and betamethasone 7 days (P = .001), but not 2 days, before delivery. Inflammation improves preterm lung function because of increases in surfactant. Antenatal corticosteroids further augment lung function through an apparently independent mechanism.

Ureaplasma parvum or Mycoplasma hominis as Sole Pathogens Cause Chorioamnionitis, Preterm Delivery, and Fetal Pneumonia in Rhesus Macaques

The authors assess causal, cellular and inflammatory links between intraamniotic infection with Ureaplasma parvum or Mycoplasma hominis and preterm labor in a nonhuman primate model. Long-term catheterized rhesus monkeys received intraamniotic inoculations of clinical isolates of Ureaplasma parvum serovar 1, M hominis, media control or physiological saline. Genital mycoplasmas were quantified in amniotic fluid (AF) and documented in fetal tissues by culture and PCR. In association with elevated AF colony counts for U parvum or M hominis, there was a sequential upregulation of AF leukocytes, proinflammatory cytokines, prostaglandin E2 and F2a, metalloproteinase-9 and uterine activity ( P< .05). Fetal membranes and lung were uniformly positive for both microorganisms; fetal blood and cerebrospinal fluid cultures and PCR were more often positive for M hominis than U parvum. Histopathologic findings of chorioamnionitis, a systemic fetal inflammatory response and pneumonitis worsen with duration of in utero infection. U parvum or M hominis, as sole pathogens, elicit a robust proinflammatory response which contributes to preterm labor and fetal lung injury.

Differences in biofilm development and antibiotic susceptibility among clinical Ureaplasma urealyticum and Ureaplasma parvum isolates

The aim of this work was to study the ability of clinical isolates of Ureaplasma spp. to form biofilms in vitro and to compare the antibiotic susceptibility of sessile cells and their planktonic counterparts. A total of nine Ureaplasma spp. isolates recovered from unrelated male patients diagnosed with urethritis or chronic prostatitis and two isolates isolated from the urine of two healthy volunteers were included. Ureaplasma species identification was performed by 16S rDNA gene amplification and sequencing. Conventional antibiotic susceptibility tests were carried out by the broth microdilution method. Biofilm susceptibility assays were performed following the method proposed by Moskowitz using 10C urea broth medium and confirming bacterial growth by colour shift of the medium. The chi(2) test was applied to analyse the statistical differences between the MIC and the minimal biofilm inhibitory concentration. Isolates were identified as Ureaplasma urealyticum serovar 7 (five isolates), U. urealyticum serovar 13 (four isolates) and Ureaplasma parvum serovar 3 (two isolates). Biofilm formation was observed in 9 out of the 11 strains studied (82%); two isolates of U. urealyticum serovar 13 were non-biofilm formers. Global resistance percentages of planktonic cells compared with sessile cells were different for erythromycin (0% versus 44%, P = 0.02), telithromycin (22% versus 77%, P = 0.02), ciprofloxacin (66% versus 100%), levofloxacin (0% versus 33%) and tetracycline (0% versus 33%). All nine biofilm-forming strains were fully susceptible to clarithromycin in both planktonic and biofilm types of growth. These results indicate that biofilm formation can protect mycoplasma cells from antibiotics and host defences, favouring their persistence in chronically infected or colonized patients while increasing resistance to antimicrobial agents. Therefore, the capacity to form biofilms by Ureaplasma spp. isolates should be considered when antibiotic treatments are required.

Experimental amniotic fluid infection in sheep: Effects of Ureaplasma parvum serovars 3 and 6 on preterm or term fetal sheep

The objective of the study was to determine the effects in late gestation of Ureaplasma parvum serovar 3 colonization and the effects, preterm, of U. parvum serovar 6. Ewes received an intraamniotic (i.a.) injection of U. parvum serovar 6 (20 x 10(6) colony-forming units [cfu]; n = 9), U. parvum serovar 3 (20 x 10(3) cfu; n = 6), vehicle (n = 10), or saline (n = 4) on day 80 of pregnancy (d). The lambs were delivered at 125 d (U. parvum serovar 6, n = 9; saline or media controls, n = 9) or 145 d (U. parvum serovar 3, n = 6; media controls, n = 5) for assessment of inflammation and lung maturation. I.a. ureaplasmas caused histologic chorioamnionitis but not preterm delivery. Fetal lung epithelium was colonized with ureaplasmas at both gestational ages, and pulmonary interleukin-8 levels had doubled in the ureaplasma-colonized animals, compared with the controls at 145 d. Surfactant levels in bronchoalveolar lavage fluid had increased 8-fold and 2.5-fold at 125 and 145 d, respectively, after ureaplasma injection. Fetal lung inflammation and altered development accompanies ureaplasma colonization, regardless of age at delivery.

Postgenomic taxonomy of human ureaplasmas -- a case study based on multiple gene sequences.

In 2000, the full genome sequence of Ureaplasma parvum (previously known as Ureaplasma urealyticum) serovar 3 was released. In 2002, after prolonged debate, it was agreed that the former U. urealyticum should be divided into two species -- U. parvum and U. urealyticum. To provide additional support for this decision and improve our understanding of the relationship between these two species, the authors studied four 'core' genes or gene clusters in ATCC reference strains of all 14 serovars of U. parvum and U. urealyticum. These 'core' regions were the rRNA gene clusters, the EF-Tu genes (tuf), urease gene clusters and multiple-banded antigen genes (mba). The known U. parvum genome sequences (GenBank accession no. NC_002162) were used as reference. DNA insertions and deletions (indels) were found in all of the gene regions studied, except tuf, but they were found only between, not within, the two species. An incidental finding was that there was inter-copy heterogeneity for rRNA gene cluster sequences. Sequence analysis (sequence heterogeneity and especially indels) of all four selected targets consistently supported the separation of human ureaplasmas into two species. Except for multiple-banded antigen, there was less heterogeneity in amino acid sequences of proteins, between species, than in the nucleic acid sequences of the corresponding genes. The degrees of heterogeneity at the 5' end of the species-specific regions of multiple-banded antigen were almost identical for both amino acid and nucleotide sequences. Analysis of the authors' results provided an interesting case study to help resolve some common problems in the use of sequence data to infer phylogenetic relationships and support taxonomic changes. It is recommended that, to avoid confusion, the new nomenclature be used for human ureaplasmas in future publications.